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- Materials and methods
The timing and source of nutritional resources allocated to reproduction have critical consequences for reproductive strategies and hence individual fitness. Allocated resources may be derived from stored reserves or current feeding (e.g. Sibly & Calow 1984; Wheeler 1996; Boggs 1997a). Organisms are therefore often categorized as ‘capital breeders’vs‘income breeders’ (e.g. Tammaru & Haukioja 1996). Nonetheless, organisms rarely are pure capital or income breeders, but rather draw nutrients to varying degrees from reserves and current feeding (e.g. Boggs 1997a; O’Brien, Boggs & Fogel 2004). Different resource types (carbohydrates, lipids, amino acids, etc.) may also be differentially drawn from reserves and current feeding (e.g. Boggs & Ross 1993; Boggs 1997b). This is particularly true for species whose diet changes with age – which includes most holometabolous insects. Given the need for resource congruence (the use of nutrient types in a specified ratio; Bazzaz 1996), storage, foraging and reproduction are linked strategies for such insects. Understanding the relative reproductive importance of stored (usually larval-derived) and current (usually adult-derived) resources, and how that importance changes through time, is thus a critical element to understanding the functional basis of reproductive patterns.
Lepidoptera are holometabolous insects that exhibit a range of reproductive and foraging strategies, and hence are useful for exploring the impact of diverse feeding habits on allocation strategies. Almost all species have herbivorous larvae, with the known exception of a few partly carnivorous lycaenids (e.g. Elmes et al. 2001). As adults, many species are strict nectivores, while others supplement their nectar diet with substrates ranging from pollen to mud, dung or carrion (e.g. Gilbert 1972; Boggs 1987; Boggs & Jackson 1991; DeVries, Murray & Lande 1997; Beck, Mühlenberg & Fiedler 1999). The nutritional composition of nectar is fairly well understood (e.g. Pacini, Nepi & Vespini 2003), and we are making progress in understanding its reproductive role in butterflies (e.g. Boggs 1997b; O’Brien, Fogel & Boggs 2002; O’Brien, Boggs & Fogel 2003). In general, nectars used by Lepidoptera are carbohydrate-rich, containing small amounts of lipids, amino acids and other compounds (see Boggs 1987 for a review). Nectar-derived carbon in particular can be used to make non-essential amino acids (O’Brien et al. 2002), and determines reproductive output in some species (Boggs & Ross 1993). Additionally, amino acid-containing nectars seem to be preferred over carbohydrate-only food sources by some species and sexes (e.g. Erhardt & Rusterholz 1998; Rusterholz & Erhardt 2000; Mevi-Schutz, Goverde & Erhardt 2003). Nonetheless, essential amino acids stored from larval feeding are likely ultimately limiting to reproduction.
Rotting fruit is also a major adult food source for a large number of Lepidoptera, particularly in the tropics (e.g. DeVries et al. 1997; DeVries & Walla 2001). Researchers have informally speculated that fruit could be a richer and more diverse source of nitrogenous compounds than is nectar; however, a recent paper suggests that amino acid concentrations in fruit are similar to those in nectar (Omura & Honda 2003). However, rotting fruit may provide yeast to fruit-feeding Lepidoptera, which is an excellent source of protein to insect frugivores (Good & Tatar 2001).
Although the impact of fruit-feeding on butterfly life histories has not been studied, a rich body of literature on frugivory in birds provides some insights on the potential nutritional quality of fruit for insects (reviewed in Levey & del Rio 2001). Fruit is similar to nectar in being fairly carbohydrate-rich and nitrogen-poor (Bosque & Pacheco 2000). Up to 25% of the nitrogen in fruit pulp can be non-proteinaceaous, meaning it has little nutritional value (Levey, Bissell & O’Keefe 2000). The protein found in fruit is unbalanced in its amino acid content (Izhaki 1998), further decreasing its nutritional value to frugivorous animals. Birds maintained exclusively on fruit diets are in negative protein balance, and lose mass (Baierlein 1996). If rotting fruit is primarily a carbon source, then we would expect allocation and life-history patterns similar to those of nectivorous Lepidoptera. However, if rotting fruit provides significant amino acid resources to fruit feeding Lepidoptera, their life history may more closely resemble those Lepidoptera that supplement their diet with amino acid resources.
The best-studied example of amino acid supplementation in nectar-feeding Lepidoptera is that of pollen-feeding Heliconius butterflies. Pollen is a nitrogen-rich food source used in addition to nectar, which completely alters the life history in comparison with related butterflies that are solely nectivores: oogenesis is continuous throughout life, rather than stopping prior to adult emergence and life span is dramatically increased (Gilbert 1972; Dunlap-Pianka, Boggs & Gilbert 1977). These butterflies supplement larval-derived essential amino acids with essential amino acids from pollen feeding to some extent (O’Brien et al. 2003).
Here we use the tropical, fruit-feeding butterfly Bicyclus anynana (Butler, 1879) to explore allocation of resources from larval herbivory and adult frugivory to egg production. We employ stable isotopes to trace dietary carbon and nitrogen, and describe allocation kinetics using a turnover model (O’Brien, Schrag & del Rio 2000). Using ad libitum fed and semistarved females, we ask two questions: is fruit an important source of carbon or nitrogen resources used in egg production and what is the relative importance of fruit in egg production in this species as compared with the importance of nectar in other lepidopteran species?
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Our results reveal that adult feeding is essential for reproduction in B. anynana: females will not oviposit without adult dietary input. This finding suggests that adult diet here is even more important than in many nectivorous butterflies, which do lay eggs if fed only water. In nectivorous species from diverse taxonomic groups studied to date, fecundity increases two- to seven-fold for females with access to sugar as opposed to only water (e.g. Pieridae, Stern & Smith 1960; Nymphalidae, Murphy, Launer & Ehrlich 1983; Lycaenidae, Hill & Pierce 1989; Satyrinae, Karlsson & Wickman 1990). The most extreme case to our knowledge is that of the lycaenid Lycaena hippothoe, which increased fecundity seven-fold given sugar as opposed to only water (Fischer & Fiedler 2001). Whether the pattern observed here is specific to B. anynana, typical of fruit-feeders or due to some other cause remains to be tested.
Likewise, the stable isotope analyses indicate that the adult diet is an important source of egg nutrients in B. anynana: approximately half the carbon came from banana. The carbon that banana provides is incorporated into developing eggs quite rapidly, as the first eggs laid already reflect a nearly even ratio of larval and adult carbon sources. Eggs laid subsequently show an additional shift toward adult dietary carbon, although the magnitude of the shift is slight. The shift in egg carbon from more larval to more adult sources is well described by a simple turnover model, similar to those applied to nectar-feeding moths and butterflies (O’Brien et al. 2000, 2003). The model reflects a physiological hypothesis: that egg carbon sources come to reflect dietary carbon to the extent that is possible, and that further adult dietary input is constrained by specific nutrient requirements. The tendency for the eggs in both experiments to plateau at identical isotopic values is consistent with this hypothesis.
The initially starved females began laying eggs within 1–2 days after adult food was provided, again indicating that adult food is quickly metabolized and incorporated into eggs. Nevertheless, the estimated starting δ13C reflected slightly more larval carbon input than the first eggs laid by continuously fed butterflies, and the eggs shifted more rapidly to the maximum contribution of adult dietary carbon. The isotopic change of eggs laid by previously starved butterflies is more than seven times more rapid than that of continuously feeding butterflies. This more rapid turnover probably reflects the depletion of larval stores in starved females due to metabolism or general maintenance. With smaller larval reserves and a similar rate of intake, turnover would be expected to occur more rapidly.
Both the percentage use of adult carbon in eggs and the C : N ratios for B. anynana eggs were within the range of those documented for nectivorous butterflies by O’Brien et al. (2004). The earlier series of nectivorous butterflies examined included species that differed in the percentage of eggs mature at adult emergence, the amount of nectar eaten as an adult, and the C : N ratio of eggs. B. anynana ecloses with no eggs mature (K. Fischer, unpublished data); when compared with such nectivorous species with similar C : N ratios, the maximal percentage of adult carbon used in egg production is the same as for the pierid Colias eurytheme and similar to the nymphalid Heliconius charitonia (O’Brien et al. 2004) and the sphingid Amphion floridensis (O’Brien et al. 2000). These data suggest that fruit may play an equivalent role to nectar in egg production, with respect to carbon utilization.
The larval and the adult diets of B. anynana contrasted in δ15N, with the adult banana diet being over 2‰ more enriched in δ15N than the larval maize diet. Egg δ15N started at a value similar to the larval diet, and declined rapidly over the first week of oviposition. These data suggest the adult diet was not providing significant nitrogen for egg manufacture, at least not at the onset of oviposition. Furthermore, it suggests that egg manufacture selectively uses the lighter isotope of nitrogen, to an increasing extent over the first week of oviposition. In a study on nectar-feeding moths, moths synthesized non-essential amino acids from adult dietary carbon, using endogenous sources of amine groups (O’Brien et al. 2002). The reaction transferring amine nitrogen between amino acids has been shown to prefer 14N, causing a shift in isotope ratio of −8‰ (Macko et al. 1986, 1987). Thus, the initial drop in egg δ15N may reflect increasing levels of non-essential amino acid synthesis from adult-derived carbon sources, using endogenous nitrogen from the larval diet. Although the adult diet in this study did contain nitrogen, it was very nitrogen poor (with a C/N of 45 : 1). Thus, the fact that egg nitrogen resembled larval diet more than the adult diet supports the idea that the adult diet is primarily a source of carbon rather than nitrogen. Banana %N was only about 1–2% (dry weight), thus, they would have to consume a great deal to make up for their nitrogen losses to oviposition (estimated to total, on average, 2·4 mg N).
Interestingly, the trend in egg δ15N shifted direction after about 1 week of oviposition. Egg δ15N increased slowly but consistently, to a value slightly higher than initial egg δ15N. This reflects an isotopic enrichment of the source of egg nitrogen, which could happen via at least two means. First, the preferential use of 14N in egg manufacture could cause the nitrogen source to become enriched in 15N. This shift will ultimately be reflected in the isotope signature of the eggs. Secondly, the accumulation of nitrogen from the adult banana diet might start to increase the δ15N of the source pool. These factors will drive the δ15N of the eggs in the same direction, so our data do not allow us to evaluate the relative merits of these two hypotheses. However, egg nitrogen follows a similar pattern in butterflies maintained on sucrose alone as adults (D. M. O’Brien, unpublished data), and thus does not require the input of banana nitrogen. Thus, the data do not clearly indicate that the adult diet provides nitrogen for egg manufacture, but neither do they rule it out in the later weeks of oviposition.
In summary, our study shows that reproduction in B. anynana depends on input from the adult fruit diet, without which no eggs are laid. Overall, egg composition is similar to that of nectivorous Lepidoptera, as is the extent to which the adult diet provides carbon for egg manufacture (O’Brien et al. 2000, 2004). However, the shift in egg carbon from larval towards an adult signature is less pronounced, because the first eggs laid already contained a large contribution of carbon from the adult diet. The only species with a comparably slight shift towards the adult diet and a high contribution of adult dietary nutrients to first-laid eggs is Heliconius charitonia, a nymphalid butterfly that feeds on nitrogen-rich pollen as well as nectar (O’Brien et al. 2004). In the experiment cited above, H. charitonia were provided sugar water only; but still drew rapidly and heavily on adult dietary input for egg manufacture. This suggests that the degree of dependence on adult income and the rapidity with which it is incorporated may be associated with the expected quality of the adult diet. If so, fruit may typically provide a richer resource for reproduction than is suggested by the data here, perhaps through the growth of yeast or other micro-organisms. Alternatively, the similarity might arise from a possible higher predictability of encountering adult food throughout much of the year in tropical regions. These issues deserve further investigation.